It has become increasingly important to medical science to be able to quantify the chemical and biochemical components of a wide variety of fluids, particularly colored aqueous biological fluids such as whole blood and urine, and biological derivatives such as serum and plasma. Such ability is important in testing for exposure to hazardous materials, intoxicants, and therapeutic drugs, and in diagnostics. In some applications, it is important for a lay person to be able to perform the test outside a laboratory environment, with rapid and accurate results. For example, diabetics must test samples of their own blood for glucose several times a day to moderate their diet and medication. The test results must be both rapidly available and accurate.
Test kits for the determination of glucose in blood are well known in the art. Such test kits often involve a test strip impregnated with one or more chemicals that react in the presence of glucose to cause a color change in the test strip. The color change in the test strip is measured by known optical detection methods. While a change in either absorption, transmittance, or reflectance of a sample can be measured, typically for glucose determinations the change in reflectance is measured. Generally, as the glucose concentration in a sample increases, the strip will generate more color and become darker. A darker test strip will give off less reflectance, such that a lower reflectance level will indicate more glucose in the sample.
The presence of red blood cells in a blood sample can interfere with the reflectance reading by reflecting light back to the detector at the same wavelength as the glucose reading is made, or by blocking the transmission of light so that all the color developed in response to glucose cannot be read, or by scattering the light. Thus some test kits require that the red blood cells be filtered out of the sample or lysed prior to applying the sample to a test strip.
One series of patents relating to the colorimetric determination of glucose in blood includes U.S. Pat. Nos. 4,935,346, 5,049,487, 5,049,394, 5,179,005, and 5,304,468, all assigned to Lifescan, Inc. of Mountain View, Calif., and all incorporated herein by reference in their entireties. The method disclosed therein involves taking a reflectance reading from one surface of an inert two-sided porous matrix. The matrix is impregnated with a reagent that will interact with the analyte to produce a light-absorbing reaction product when the fluid being analyzed is applied to the first surface and migrates through the matrix to the second surface. Reflectance measurements of the second surface are made at two separate wavelengths in order to compensate for interference from hematocrit variation, and for variation caused by the chromatography of the color generated in response to glucose at a given concentration. A timing circuit is triggered by an initial decrease in reflectance caused by the wetting of the second surface by the fluid which passes through the inert matrix. The method does not require the separation of red blood cells from serum or plasma.
U.S. Pat. No. 5,484,708 assigned to Boehringer Mannheim GmbH, entitled “Method for the Colorimetric Determination of an Analyte with a PQQ-Dependent Dehydrogenase” discloses a method for the colorimetric determination of an analyte by means of enzymatic oxidation with a PQQ-dependent dehydrogenase in the presence of an electron acceptor from the group of the electron-rich aromatic nitroso compounds by enzymatic reduction of the nitroso compound to an imino compound and detection of the imino compound by color formation.
U.S. Pat. No. 5,789,255, also assigned to Lifescan, Inc. and incorporated herein by reference in its entirety, is entitled “Blood Glucose Strip having Reduced Sensitivity to Hematocrit.” This patent discloses a reagent strip comprising an anisotropic membrane having a sample side with relatively large pores and a testing side with relatively small pores. A test sample is applied to the sample side and passes through the membrane toward the testing side, while the relatively large red blood cells are filtered out of the blood sample. The membrane is impregnated with a testing agent comprising a component that reacts with glucose and oxygen to create hydrogen peroxide, a color indicator that reacts with the hydrogen peroxide, and an acrylic acid-polymer that reduces the effect of the red blood cells on the glucose concentration measurement. It is believed that the acrylic acid polymers used are of relatively high molecular weight, and affect the viscosity and flow of the sample.
In such prior art methods of testing for glucose using optical detection methods, it is recognized that the detection system must adjust for the opacity of a reflective surface. It is known that a reaction surface that is more opaque will be less sensitive to the presence of red blood cells. In some such methods, the opacity is adjusted by the use of scattering centers incorporated into the reflective surface of the matrix. Such known scattering centers include titanium dioxide, diatomaceous earth, powdered metals, minerals, and various combinations of these and other materials. Other factors known to affect opacity include pore size, pore structure, and the surface area of the supporting media.
It is one object of the invention to provide a method for testing a sample of a fluid that does not require the separation or lysing of red blood cells from a whole blood sample.
It is a further object of the invention to provide such a method that can be done with a very small sample of fluid, typically less than one microliter.
It is still another object of the invention to provide such a test method that is not sensitive to variations of hematocrit from sample to sample.
It is yet another object of the invention to provide a test strip suitable for use with the inventive method.